Stabilised solid compositions of factor VII polypeptides

ABSTRACT

The invention relates to chemically as well as physically stable compositions comprising Factor VII or a Factor VII-related polypeptide such that these compositions can be stored, handled and used at room temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. application Ser.No. 10/609,780 filed Jun. 30, 2003 which claims priority under 35 U.S.C.120 of international application no. PCT/DK03/00419 filed Jun. 20, 2003,and claims priority under 35 U.S.C. 119 of Danish application no. PA2002 00963 filed Jun. 21, 2002 and U.S. application No. 60/394,153 filedJul. 3, 2002, and, the contents of which are fully incorporated hereinby reference.

FIELD OF INVENTION

The present invention relates to chemically as well as physically stablecompositions comprising Factor VII or a Factor VII-related polypeptidesuch that these compositions can be stored, handled and used at roomtemperature.

BACKGROUND OF THE INVENTION

Factor VII is a polypeptide involved in the blood clotting process.Today, Factor VIIa can be made by recombinant techniques (rFVIIa) and iswidely used as a pro-haemostatic agent. Factor VII (human wild-type) hasbeen described in U.S. Pat. No. 4,784,950. rFVIIa offers today a rapidand highly effective pro-haemostatic response in haemophilic individualsexperiencing bleeding. Advantageously, rFVIIa can be used for treatinghaemophilic individuals that cannot be treated with other coagulationfactor products due to antibody formation. Also individuals sufferingfrom Factor VII deficiency or individuals having a normal coagulationsystem but still experiencing excessive bleeding can be treatedsuccessfully with rFVIIa.

Several parameters need to be considered when developing a medicamentcomprising a polypeptide such as, e.g., Factor VIIa. By example, themedicament needs to be effective, safe and lead to good patientcompliance. Moreover, the medicament may be formulated for parenteraladministration using pharmaceutically acceptable excipients, which willhave to meet with the approval of various world-wide medical regulatoryagencies. For the purpose of parenteral administration, it is highlydesirable that the formulation is approximately isotonic and that the pHof the formulation is in a physiologically suitable range uponinjection/infusion, otherwise it may result in pain and discomfort forthe patient. For a general review of protein formulations, see, forexample, Cleland et al.: The development of stable protein formulations:A closer look at protein aggregation, deamidation and oxidation,Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10(4):307-377; and Wang et al., Parenteral formulations of proteins andpeptides: Stability and stabilizers, Journal of Parenteral Science andTechnology 1988 (Supplement), 42 (2S).

However, for medicaments comprising polypeptides the safety may directlybe related to the physical and chemical stability of the polypeptide.Being a polypeptide, Factor VII or a Factor VII-related polypeptide issusceptible to physical degradation, including denaturation andaggregation such as the formation of soluble or insoluble aggregates inthe form of dimers, oligomers and polymers, or to chemical degradation,including for example, hydrolysis, deamidation and oxidation.Consequently, the said physical and chemical instability may lead toloss of activity of the Factor VII polypeptide, formation of toxic andimmunogenic degradation products, serious risk of introducing thrombosisupon injection of the degraded Factor VII polypeptides, clogging ofneedles used for injections and risk of non-homogeneity, to name just afew.

Thus, it is essential to provide compositions comprising Factor VIIpolypeptides that is stabilised against physical and chemicaldegradation.

Today, recombinantly-made FVII polypeptide is provided as freeze-driedproduct that is meant to be stored at temperatures between about 2 andabout 8° C. The requirement of cooled conditions causes a burden to andis inconvenient for the manufacturer or provider as well as the end user(the patient).

The actual recombinantly-made FVII product is NovoSeven® (Novo NordiskA/S, Denmark) that consists of 1.2 mg recombinant human Factor VIIa,5.84 mg NaCl, 2.94 mg CaCl₂, 2H₂O, 2.64 mg Glycylglycine, 0.14 mgpolysorbate 80 and 60.0 mg mannitol. When reconstituted by 2.0 ml ofwater for injection (WFI), the pH is 5.5 and the thus preparedFVII-containing solution is sufficiently stable for 24 hours at roomtemperature.

The present investigators have found that upon storage of thelyophilised NovoSeven® product for 6 months at 25° C. about 6 to 7% w/wof the initial content of the rFVIIa is present in the form ofaggregates.

Thus, compositions comprising Factor VII polypeptides need to bestabilised so as allowing storage and handling at ambient temperatures.However, the instability of polypeptides relates to several parametersand it is impossible to predict the proper manner of stabilising aFactor VIIa or a Factor VII-related polypeptide.

One approach of stabilising a protein relates to removal of water fromthe protein, e.g. such as providing the protein in the form of alyophilised cake, the final matter obtained in a freeze-drying process.However, the freeze-drying process itself is also harmful to proteins;during freeze-drying, the protein solution is first cooled untiladequately frozen and bulk water in the protein solution will form iceat this stage. The protein is hereby prone to freeze-induced stressresulting in deformation and precipitation. In the next step, theso-called primary drying stage, the ice sublimes and in the secondarydrying stage, adsorbed or bound water is removed under elevatedtemperatures. During this water removal, the proteins may loose theirproper conformation that is provided mainly through hydrogen bonding.

Therefore, to preserve protein conformation, activity and stabilityduring freeze-drying, the polypeptide solution needs to be supplementedwith sufficient amounts of proper excipients with cryoprotectant and/orlyoprotectant properties so as to protect the protein fromfreeze-induced stress and/or stress during removal of water,respectively.

Additionally, when providing a lyophilised product, an essential featurerelates to the properties of the lyophilised cake. It needs to have goodproperties as to its form and structure, i.e. it should not collapse inthat such collapsed cakes can be hard or even impossible to dissolve(reconstitute) before use. Conversely, the physical structure of thelyophilised cake may not be too loosen and soft. Therefore, one or moreso-called bulking agents are added to the protein solution beforefreeze-drying.

Other publications of interest regarding stabilisation of polypeptidesare as follows:

U.S. Pat. No. 2,001,0031721 A1 (American Home Products) concerns highlyconcentrated, lyophilised, and liquid Factor IX formulations.

WO 97/26909 (Genetics Institute) concerns lyophilised preparations ofFactor IX suitable for storage and administration. The preparations maycomprise sucrose or mannitol as a cryoprotectant.

WO 95/28954 (Genetics Institute) concerns preparations of Factor IXsuitable for storage and administration. The preparations may comprisesucrose as a cryoprotectant.

It is an objective of the present invention to provide stablecompositions of Factor VII polypeptides, substantially without thepresence of degradation products and without decreased activity of theFactor VII polypeptides, preferable after prolonged storage at ambientconditions, e.g. for at least 6 months. Furthermore, it is an objectivethat the stable compositions are suitable for parenteral administrationso as not to cause any inconvenience for the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the stability of the six formulations that weretested as described in Example 11.

FIGS. 2A and 2B show the contents of aggregate forms for dimmer,oligomer, and oxidized forms of the formulations of the invention.

SUMMARY OF THE INVENTION

It has been found by the present investigators that Factor VIIpolypeptides can be provided in a composition that is sufficient stableso as to allow for storage at room temperature for about at least 8months. The investigators have found that the stabilisation relates tothe proper combining of some pharmaceutically acceptable excipients.

Accordingly, the present invention relates in a first aspect tostabilised compositions that have a moisture content of not more thanabout 3% and comprises a Factor VII polypeptide and at least onestability agent selected from the group consisting of a) to e):

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine.

In a further aspect, the invention relates to a method of preparing astable Factor VII polypeptide comprising the steps of:

i) Providing said Factor VII polypeptide in a solution comprising atleast one stability agent selected from the group consisting of a) toe):

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine;

ii) Processing said solution so as to obtain a solid composition with amoisture content of not more than about 3% w/w.

As mentioned, stabilised Factor VII polypeptides are requested so as tominimise the risk of adverse events and to improve safety and efficacywhen administering Factor VII polypeptides for therapeutic purposes.Therefore, a still further aspect of the invention relates to the use ofFactor VII polypeptide for the preparation of a medicament for treatinga Factor VII-responsive syndrome, said medicament comprising acomposition comprising; a Factor VII polypeptide and a at least onestability agent selected from the group consisting of

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine,

said composition having a moisture content of not more than about 3%.

Finally, the invention relates to administering said Factor VIIpolypeptides for treating a Factor VII-responsive syndrome comprisingadministering to a subject in need thereof an effective amount of acomposition comprising a Factor VII polypeptide and at least onestability agent selected from the group consisting of

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine;

said composition having a moisture content of not more than about 3%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to storage-stable compositions comprisingFactor VII polypeptides. The compositions can be stored at roomtemperature for an extended period of time without causing substantialdegradation of the Factor VII polypeptide. By room temperature is meantthe ambient temperature inside a room; it normally ranges from about 5°C. to about 40° C., such as from about 10° C. to 30° C., or 15° C. to25° C.

By proper predetermined combination of particular pharmaceuticallyacceptable excipients, the present investigators have providedstabilised compositions comprising Factor VII polypeptides, thusallowing the compositions to be stored at room temperature for anextended period of time such as at least about 8 months. Advantageously,the stabilised compositions need not to be stored at cooled conditions,such as between 2 and 8° C.

The present invention also concerns storage-stable compositions that arestable for at least about 8 months upon storage at about 30° C. Thecomposition is preferably stored in the dark. Thus, the presentinvention makes it possible to store such compositions at roomtemperature without increasing the risk of adverse events to the patientadministering such compositions. Advantageously, the improvedstorage-stability will also result in reduced cost in that no specialcooled conditions are required upon storage, further resulting in moreconvenient handling of the composition by the user.

The term “Factor VII polypeptide” is denoted to mean any Factor VIIpolypeptide that is effective in preventing or treating bleeding. Thisincludes Factor VII polypeptides derived from blood or plasma, orproduced by recombinant means.

As used herein, “Factor VII polypeptide” encompasses, withoutlimitation, Factor VII, as well as Factor VII-related polypeptides. Theterm “Factor VII” is intended to encompass, without limitation,polypeptides having the amino acid sequence 1-406 of wild-type humanFactor VII (as disclosed in U.S. Pat. No. 4,784,950), as well aswild-type Factor VII derived from other species, such as, e.g., bovine,porcine, canine, murine, and salmon, said Factor VII derived from bloodor plasma, or produced by recombinant means. It further encompassesnatural allelic variations of Factor VII that may exist and occur fromone individual to another. Also, the degree and location ofglycosylation or other post-translation modifications may vary dependingon the chosen host cells and the nature of the host cellularenvironment. The term “Factor VII” is also intended to encompass FactorVII polypeptides in their uncleaved (zymogen) form, as well as thosethat have been proteolytically processed to yield their respectivebioactive forms, which may be designated Factor VIIa. Typically, FactorVII is cleaved between residues 152 and 153 to yield Factor VIIa.

As mentioned, the term “Factor VII polypeptides” is also denoted to mean“Factor VII-related polypeptides” The term “Factor VII-relatedpolypeptides” are intended to encompass such polypeptides in theiruncleaved (zymogen) form, as well as those that have beenproteolytically processed to yield their respective bioactive forms. Asused herein, “Factor VII-related polypeptides” encompass, withoutlimitation, polypeptides exhibiting substantially the same or improvedbiological activity relative to wild-type human Factor VII. Thesepolypeptides include, without limitation, Factor VII or Factor VIIa thathas been chemically modified and Factor VII variants into which specificamino acid sequence alterations have been introduced that slightlymodify or improve the biological activity of the polypeptide.

Moreover, Factor VII-related polypeptides, including variants of FactorVII exhibiting substantially the same or better biological activity thanwild-type Factor VII, include without limitation polypeptides having anamino acid sequence that differs from the sequence of wild-type FactorVII by insertion, deletion, or substitution of one or more amino acids.

Factor VII-related polypeptides, including variants, havingsubstantially the same or improved biological activity relative towild-type Factor VIIa encompass those that exhibit at least about 25%,preferably at least about 50%, more preferably at least about 75%, morepreferably at least about 100%, more preferably at least about 110%,more preferably at least about 120%, and most preferably at least about130% of the specific activity of wild-type Factor VIIa that has beenproduced in the same cell type, when tested in one or more of a clottingassay, proteolysis assay, or TF binding assay as described in thepresent specification.

In some embodiments the Factor VII polypeptides are Factor VII-relatedpolypeptides, in particular variants, wherein the ratio between theactivity of said Factor VII polypeptide and the activity of native humanFactor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the“In Vitro Hydrolysis Assay” (see Example 9, below); in otherembodiments, the ratio is at least about 2.0; in further embodiments,the ratio is at least about 4.0. In some embodiments of the invention,the Factor VII polypeptides are Factor VII-related polypeptides, inparticular variants, wherein the ratio between the activity of saidFactor VII polypeptide and the activity of native human Factor VIIa(wild-type FVIIa) is at least about 1.25 when tested in the “In VitroProteolysis Assay” (see Example 9, below); in other embodiments, theratio is at least about 2.0; in further embodiments, the ratio is atleast about 4.0; in further embodiments, the ratio is at least about8.0.

Non-limiting examples of Factor VII variants having substantially thesame or improved biological activity as wild-type Factor VII includeS52A-FVII, S60A-FVII (Lino et al., Arch. Biochem. Biophys. 352: 182-192,1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I -FVII, L305T-FVII,F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII,M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII,V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII,V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII,E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII;FVIIa variants exhibiting increased proteolytic stability as disclosedin U.S. Pat. No. 5,580,560; Factor VIIa that has been proteolyticallycleaved between residues 290 and 291 or between residues 315 and 316(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); oxidized formsof Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54,1999); FVII variants as disclosed in PCT/DK02/00189; FVII variantsexhibiting increased proteolytic stability as disclosed in WO 02/38162(Scripps Research Institute); FVII variants having a modified Gla-domainand exhibiting an enhanced membrane binding as disclosed in WO 99/20767(University of Minnesota); FVII variants as disclosed in WO 01/58935(Maxygen ApS); FVII variants having increased biological activitycompared to wild-type FVIIa as disclosed in WO 01/83725, WO 02/22776, WO02/077218, PCT/DK02/00635, Danish patent application PA 2002 01423,Danish patent application PA 2001 01627; WO 02/38162 (Scripps ResearchInstitute); and FVIIa variants with enhanced activity as disclosed in JP2001061479 (Chemo-Sero-Therapeutic Res Inst.).

Examples of factor VII or factor VII-related polypeptides include,without limitation, wild-type Factor VII, L305V-FVII,L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII,V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII, L305V/V158D-FVII,L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V158T-FVII,L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII,L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII,L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII,L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII,L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVII,L305V/V158D/E296V/M298Q/K337A-FVII, L305V/V158T/E296V/M298Q/K337A-FVII,S314E/K316H-FVII, S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/V158T-FVII,K316H/L305V-FVII, K316H/K337A-FVII, K316H/V158D-FVII, K316H/E296V-FVII,K316H/M298Q-FVII, K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII, K316Q/V158T-FVII,S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII,S314E/L305V/M298Q-FVII, S314E/L305V/V158T-FVII,S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVII,S314E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVII,S314E/L305V/V158D/M298Q-FVII, S314E/L305V/V158D/E296V-FVII,S314E/L305V/V158T/M298Q-FVII, S314E/L305V/V158T/E296V-FVII,S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296V/M298Q-FVII,S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII,S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII,S314E/L305V/V158D/E296V/K337A-FVII,S314E/L305V/V158D/E296V/M298Q/K337A-FVII,S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII,K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII,K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII,K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII,K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII,K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII,K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII,K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII,K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A-FVII,K316H/L305V/V158D/E296V/M298Q/K337A-FVII,K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII,K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII,K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII,K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII,K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII,K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII,K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII,K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII,K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A-FVII,K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII,F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII,F374Y/L305V/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII,F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII,F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII,F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII,F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII,F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII,F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII,F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII,F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII,F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII,F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII,F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII,F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII,F374Y/K337A/V158T/E296V-FVII, F374Y/K337A/M298Q/E296V-FVII,F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII,F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII,F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII,F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-FVII,F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A-FVII, F374Y/L305V/E296V/M298Q/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII,F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII,F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII,F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII,F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII,F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII,F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII,F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII,F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII,F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,F374Y/L305V/E296V/K337A/V158T/S314E-FVII,F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,F374Y/L305V/V158D/E296V/K337A/S314E-FVII,F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,S60A-Factor VII; and P11Q/K33E-FVII, T106N-FVII, K143N/N145T-FVII,V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII,K143N/N145T/R315N/V317T-FVII; FVII having substitutions, additions ordeletions in the amino acid sequence from 233Thr to 240Asn, FVII havingsubstitutions, additions or deletions in the amino acid sequence from304Arg to 329Cys, and FVII having substitutions, deletions, or additionsin the amino acid sequence Ile153-Arg223.

For purposes of the invention, biological activity of Factor VIIpolypeptides (“Factor VII biological activity”) may be quantified bymeasuring the ability of a preparation to promote blood clotting usingFactor VII-deficient plasma and thromboplastin, as described, e.g., inU.S. Pat. No. 5,997,864. In this assay, biological activity is expressedas the reduction in clotting time relative to a control sample and isconverted to “Factor VII units” by comparison with a pooled human serumstandard containing 1 unit/ml Factor VII activity. Alternatively, FactorVIIa biological activity may be quantified by

-   -   (i) Measuring the ability of Factor VIIa or a Factor        VIIa-related polypeptide to produce activated Factor X (Factor        Xa) in a system comprising TF embedded in a lipid membrane and        Factor X. (Persson et al., J. Biol. Chem. 272:19919-19924,        1997);    -   (ii) Measuring Factor X hydrolysis in an aqueous system (“In        Vitro Proteolysis Assay”, see Example 12, below);    -   (iii) Measuring the physical binding of Factor VIIa or a Factor        VIIa-related polypeptide to TF using an instrument based on        surface plasmon resonance (Persson, FEBS Letts. 413:359-363,        1997); and    -   (iv) Measuring hydrolysis of a synthetic substrate by Factor        VIIa and/or a Factor VIIa-related polypeptide (“In Vitro        Hydrolysis Assay”, see Example 12, below); and    -   (v) Measuring generation of thrombin in a TF-independent in        vitro system.

The term “Factor VII biological activity” or “Factor VII activity” isintended to include the ability to generate thrombin; the term alsoincludes the ability to generate thrombin on the surface of activatedplatelets in the absence of tissue Factor.

Example 12 of the present specification describes in detail assaysuseful for assaying FVII biological activity.

Moreover, throughout the present specification, the terms below have thefollowing meaning:

The term “stabilising” is intended to encompass minimising the formationof aggregates (insoluble and/or soluble) and/or chemical degradation aswell as providing maintenance of pH and proper conformation of theprotein during storage or production of the compositions so thatsubstantial retention of biological activity and protein stability ismaintained. Moreover, stabilising is also denoted to mean lyoprotectionand cryoprotection of the protein during production of the compositionsat freeze-drying conditions.

The term “structural stabilisation” or “structural stability” isintended to encompass the ability to form a lyophilised plug or cakewith good properties and looks, e.g. such that it does not collapse andis readily dissolved before use.

The term “storage-stable” is intended to define a product that isstabilised upon storage at temperatures between 5° C.-40° C. and remainswithin pre-selected product specifications for a suitable timeperiod—often several months.

The term “physical stability” of Factor VII polypeptides relates to theformation of insoluble and/or soluble aggregates in the form of dimeric,oligomeric and polymeric forms of Factor VII polypeptides as well as anystructural deformation and denaturation of the molecule.

The term “chemical stability” is intended to relate to the formation ofany chemical change in the Factor VII polypeptides upon storage indissolved or solid state at accelerated conditions. By example arehydrolysis, deamidation and oxidation. In particular, thesulphur-containing amino acids are prone to oxidation with the formationof the corresponding sulphoxides.

The term “cryoprotectants” as used herein generally include agents,which provide stability to the protein from freezing-induced stresses.Examples of cryoprotectants include polyols such as, for example,mannitol, and include saccharides such as, for example, sucrose, as wellas including surfactants such as, for example, polysorbate, poloxamer orpolyethylene glycol, and the like. Cryoprotectants also contribute tothe tonicity of the formulations.

The term “lyoprotectant” as used herein includes agents that providestability to the protein during water removal upon the drying process ofthe lyophilisation process. For example by maintaining the properconformation of the protein. Examples of lyoprotectants includesaccharides, in particular di- or trisaccharides. Cryoprotectants mayalso have lyoprotectant effects.

The term “agent suitable for keeping the pH in the range of 3 to 9”encompasses those agents that maintain the solution pH in an acceptablerange between 3.0 and 9.0. Typical examples of agents capable of keepingthe pH within a range of 3 to 9 are the acid form or salts of citricacid, acetic acid, histidine, malic acid, phosphoric acid, tartaricacid, succinic acid, MES, HEPES, PIPES, imidazole, TRIS, lactic acid,glutaric acid and glycylglycine. It is to be understood that acombination of agents, wherein the combination of agents is suitable formaintaining the pH in the above-described range, may also be used in thepresent invention.

The term “lyophilised cake” as used herein is denoted to encompass thesolid composition obtained upon processing a dissolved or at least apartly dissolved composition under conditions involving at least onestep of cooling said dissolved/partly dissolved composition to icefollowed by at least one step of vacuum drying.

The term “lyophilization” and “freeze-drying” encompasses a processduring which liquid is removed from a dissolved or at least partlydissolved composition under conditions involving at least one step ofcooling the dissolved or partly dissolved solution to ice followed byvacuum drying. Lyophilization, or freeze-drying, is the most commonprocess for making solid protein pharmaceuticals. The process consistsof two major steps: freezing of a protein solution, and drying of thefrozen solid under vacuum. The drying step is further divided into twophases: primary and secondary drying. The primary drying removes thefrozen water (sublimation of ice) and the secondary drying removes thenon-frozen “bound” water (desorption of water). More detailed analysisof each lyophilization step is provided in, e.g., Wang et al,International Journal of Pharmaceutics 203 (2000): 1-60 (see section 4,page 16 ff.).

Typically, a composition is freeze-dried by filling into vials, freezingon the shelves of the freeze-dryer, after which a vacuum is establishedand the shelves heated to implement primary drying (or sublimation ofice). Thereafter, secondary drying (or desorption of sorbed water) takesplace at a higher temperature until the completion of the process, i.e.,where the composition contains a sufficiently low content of moisture(water). Methods for freeze-drying are generally known in the art, see,for example, Wang et al, International Journal of Pharmaceutics 203(2000): 1-60.

It is within the ordinary skill of the practitioner to optimize thefreeze-drying conditions in regard of temperature(s), time(s) at eachtemperature, and also pressure that is to be used during the process fora specific composition.

The term “moisture content” is meant to encompass water associated withthe product, including, without limitation, water in adsorbed form, suchas unfrozen water entrapped in or adsorbed to the frozen solute phaseand/or associated with the amorphous phase or adsorbed to thecrystalline solid. The term “water content” is used interchangeably with“moisture content”. The desired residual moisture level (moisturecontent) is a function of the duration and the temperature of thesecondary drying step. Several methods for determining the residualmoisture content during lyophilization are known in the art; forexample, an electronic hygrometer or a residual gas analyser may beused. Moisture contents of freeze-dried formulations can be determinedby several methods known in the art, such as, for example,loss-on-drying, Karl Fischer titration, thermal gravimetric analysis(TGA), gas chromatography (GC), or near IR (see, e.g. Wang et al,International Journal of Pharmaceutics 203 (2000): 1-60). Methods fordetermining water contents (moisture contents) are also described inboth the European and U.S. Pharmacopoeias. For example, determination ofwater content can be performed by Karl Fischer coulometric titration asdescribed in the U.S. Pharmacopoeia (USP <921, Ic>) or the EuropeanPhamacopoeia (EP <2.5.32>).

In brief, the method is as follows:

Determination of water content by coulometric titration: The KarlFischer reaction is used in the coulometric determination of water basedupon the quantitative reaction of water with sulphur dioxide and iodinein an anhydrous medium. Iodine is produced electrochemically in thereaction cell by oxidation of iodide. The iodine produced at the anodereacts immediately with the water and the sulphur dioxide contained inthe reaction cell. The amount of water in the substance is directlyproportional to the quantity of electricity up until the titrationend-point. When all of the water in the cell has been consumed, theend-point is reached and thus an excess of iodine appears which isdetected electrometrically thus indicating the end-point. The percentagewater content present in the substance is then calculated.

Moisture content may be defined in terms of the weight of the sample inthe vial at the time of analysis (i.e. solids plus the waterpresent—called wet weight basis) or it may be defined in terms where itis corrected for the measured water in the sample (i.e. dry weightbasis). In case of freeze-dried products with low moisture contents thetwo measurements (wet weight basis vs. dry weight basis) yield verysimilar results. As used herein, moisture contents are defined in termsof the solids plus the water present (i.e., wet weight basis).

The term “bulking agent” generally includes agents, which provide goodlyophilised cake properties, which form a pharmaceutically elegantproduct, which help the protein overcome various stresses,shear/freezing for example, associated with lyophilisation processes,and which help to maintain protein activity levels during thefreeze-drying process and subsequent storage. Typical examples ofbulking agents include mannitol, glycine, sucrose, lactose. These agentsmay also contribute to the tonicity of the formulations.

The term “tonicity modifier” is denoted to mean any agent capable ofadjusting the tonicity of the composition such that upon dissolving thecomposition at the time of use, the composition has a tonicity withinthe physiological range of the blood, peritoneal fluid or other relevantbody fluids. Obviously, the tonicity may also depend on whether thereconstitution solution comprises tonicity-modifying agents.

The term “surfactants” generally include those agents, which protect theprotein from air/solution interface-induced stresses andsolution/surface induced-stresses. For example surfactants may protectthe protein from aggregation. Suitable surfactants may include e.g.polysorbates, polyoxyethylene alkyl ethers such as Brij 35®, orpoloxamer such as Tween 20, Tween 80, or poloxamer 188. Preferreddetergents are poloxamers, e.g. Poloxamer 188, Poloxamer 407;polyoxyethylene alkyl ethers, e.g. Brij 35®, Cremophor A25, SympatensALM/230; and polysorbates/Tweens, e.g. Polysorbate 20, Polysorbate 80.More preferred are Poloxamers, e.g. Poloxamer 188, and Tweens, e.g.Tween 20 and Tween 80.

The term “initial content” relates to the amount of Factor VIIpolypeptides added to a composition at the time of preparation. Theconcentration given herein (mg/ml) refer to either the concentration inthe solution of Factor VII polypeptide before removing the moisture(e.g. before freeze-drying) or in the reconstituted composition, or isreferred as % w/w, which then relates to the concentration in the solidcomposition, e.g. the lyophilised cake.

As used herein, amounts specified are understood to be ± about 10%; thusabout 50 mM includes 50 mM±5 mM, 4% includes 4%±0.4%, etc.

As stated above, the present investigators have contributed essentiallyto the art by stabilising Factor VII polypeptides thereby allowinglong-term storage without causing increased risk and inconvenience tothe user.

The present investigators have found that a number of crucial parametersneed to be adjusted in stabilising Factor VII polypeptides. Oneimportant parameter relates, at least in part, to the moisture content,e.g. water. The moisture content should be limited. As a furtheressential parameter, the composition should at least include onestability agent. According to the present invention, a proper stabilityagent includes the combination of at least two groups ofpharmaceutically acceptable excipients selected from the groupconsisting of antioxidants, saccharides and polyols. The saccharides andpolyols have lyoprotectant and/or cryoprotectant properties that may beimportant, at least in part, in the event where the composition isfreeze-dried. In general, improved stability may be achieved, in part,by the proper combination of at least two of these groups of excipients.However, more specifically it was found that when said combinationcomprises a saccharide (sucrose) or an antioxidant (methionine), thestabilising effect may be even more significant. Moreover, it was alsosurprisingly found that methionine prevents oxidative degradation of theFactor VII polypeptides.

Hence in a first aspect the invention relates to a compositioncomprising a Factor VII polypeptide and at least one stability agentselected from the group consisting of

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine,

said composition having a moisture content of not more than about 3%.

That is to say that one embodiment of the invention comprises acombination of an antioxidant and mannitol; a second embodimentcomprises a combination of methionine and a polyol; another embodimentcomprises a combination of a saccharide and mannitol; in still anotherembodiment, the composition comprises a combination of sucrose and apolyol; and finally in another suitable embodiment, the compositioncomprises methionine.

As stated, the stabilising agent according to the invention includescombining at least two groups of pharmaceutically acceptable excipients.In suitable embodiments thereof, the stabilising agent further comprisesa third group of excipients. Hence, in one embodiment, the combinationof an antioxidant and mannitol further comprises a saccharide. In asecond embodiment thereof, the combination of methionine and a polyolfurther comprises a saccharide. In still interesting embodiments, thecombination of a saccharide and mannitol further comprises anantioxidant and the combination of sucrose and a polyol furthercomprises an antioxidant. In one embodiment, the composition of theinvention comprises mannitol and sucrose; in another embodiment, thecomposition comprises mannitol, sucrose and methionine; in anotherembodiment, the composition comprises mannitol and trehalose; in anotherembodiment, the composition comprises mannitol, trehalose and methionine

According to the invention, the Factor VII polypeptide is meant toencompass the polypeptides as described above. In suitable embodimentsof the invention, the Factor VII polypeptide is selected from the groupconsisting of Human Factor VIIa, Recombinant Human Factor VIIa and aFactor VII Sequence Variant. Preferably, the Factor VII Polypeptide isHuman Factor VIIa or Recombinant Human Factor VIIa or a FactorVII-related polypeptide wherein the ratio between the activity of saidFactor VII-related polypeptide and wild-type Factor VII is at least 1.25when tested in one or more of the “In Vitro Proteolysis Assay” and the“in Vitro Hydrolysis Assay” as described in the present specification.

As stated, the moisture content should be limited. For the purposes ofthe present invention, the Factor VII polypeptides, when provided inbulk, may be provided in solid or liquid form. However, typically theFactor VII polypeptides, when provided in bulk, are in liquid form.Thus, further processing of the bulk proteins for the manufacturing ofcompositions requires the steps of adding suitable excipients andremoving the liquid from the bulk, said addition of excipients may becarried out before or after removing the liquid. One such mean forremoving liquid from a protein relates to freeze-drying. Therefore, in apreferred embodiment of the present invention, the composition is in theform of a lyophilised cake. However, the present invention does notpreclude other processes that are suitable for removing the liquid fromthe bulk polypeptide so as to achieve a solid composition with moisturecontent of not more than about 3% w/w.

Moreover, according to the invention, the moisture content is preferablynot more than about 2.5% w/w, preferably not more than about 2% w/w,most preferably not more than about 1.5% w/w.

As may be understood, the invention relates, in part, to limiting thedegradation of Factor VII polypeptides during preparation, e.g. duringadmixing of excipients and removing of liquid so as to achieve a solidcomposition with moisture content of the most 3% w/w, and to limitingsaid degradation from the time of manufacturing the solid compositionuntil the time of use, e.g. until the time when the composition is to beadministered by a patient.

Therefore, as a still further parameter in stabilising compositionscomprising Factor VII polypeptides, the pH should be kept in the pHrange within 3 to 9 when dissolved in aqueous solvent, such as, e.g.,pure water or aqueous buffer. That is to say that the pH in thepolypeptide solution at the time before removing the moisture content,e.g. before freeze-drying, should be kept within a pH of about 3 toabout 9. Advantageously, this pH range is also within the desiredphysiological range, thereby causing no harm to the user uponadministering the composition by parenteral means. Preferably, the pH ofthe solution is from about 4.0 to about 9.0, such as 4.0 to 8.0, 4.0 to7.5, 4.0 to 7.0, 4.5 to 7.0, 4.5 to 6.8, 4.5 to 6.5, 5.0 to 7.0, 5.0 to6.5, 5.0 to 6.0, 5.5 to 6.5, or about 5.5 to about 6.0 such as about5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

Hence, interesting embodiments of the invention further comprises anagent suitable for keeping the pH of said composition in the range of 3to 9 when dissolved in aqueous solution (e.g. water). Accordingly, insuitable embodiments thereof, the agent suitable for keeping the pH inthe range of 3 to 9 is selected from the group consisting of acid orsalts of citric acid, acetic acid, histidine, malic acid, phosphoricacid, tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, lacticacid, glutaric acid, PIPES and glycylglycine.

Furthermore, the suitable agent for keeping the pH in the range of 3 to9 may also be a mixture of at least two such listed agents, wherein themixture is able to provide a pH value in the specified range. Theconcentration of the suitable agents is in the range of from about 0.1mM to 100 mM; from about 0.2 mM to 50 mM; from about 0.5 mM to 25 mM;from about 1 mM to 20 mM; or from about 1 mM to 10 mM.

As can be seen from Example 5 and 6, the present investigators haveprovided compositions with low contents of oxidised forms and aggregatesupon termination of the manufacturing process, i.e. upon termination ofthe freeze-drying process, by combining proper amounts of mannitol (apolyol), sucrose (a saccharide) and an antioxidant (methionine). Thus,the compositions according to the invention are characterised by havinga low initial content of oxidised forms and aggregates before beingsubjected to storage.

Degradation of the Factor VII polypeptide by the oxidative pathway aswell as by the aggregation pathway are sensitive parameters ofstability.

Typically, the compositions are stabilised upon termination of thefreeze-drying such that less than 5% w/w, such as less than 4, 3 or 20%w/w of the initial content of Factor VII polypeptide is converted intoits oxidised forms. The initial content of said Factor VII polypeptidebeing the amount added to the composition upon preparation of thecomposition before the freeze-drying step. Moreover, less than 5% w/w,such as less than 4.0%, 3.0%, 2.5%, 2%, 1.5%, or less than 1% w/w of theinitial content of Factor VII polypeptide is recovered as aggregateforms, as determined by conventional analytical methods (such as, forexample, as described in the Examples of the present application).

The present investigators have found that further degradation (i.e., ascalculated from the time of termination of the manufacturing processuntil 8 months of storage at 30° C.) of a Factor VII polypeptide isminimal upon storage under ambient conditions. As can be seen fromExample 5, upon storage at 30° C. for 8 months the increase is such thatless than 10% w/w of the initial content of Factor VII polypeptides isrecovered as oxidised forms of Factor VII polypeptides in addition tothe content of said oxidised forms present at the time of terminationfreeze-drying. Of great importance, it was found that compositionscomprising an antioxidant (methionine) are more stable towards oxidativedegradation of the Factor VII polypeptide.

Therefore, according to the present invention, suitable compositionshave a limited increase in the content of oxidised forms upon storagefor at least 8 months at ambient conditions. That is to say that instill more interesting embodiments, the composition is stable such thatno more than about 6% w/w of the initial content of Factor VIIpolypeptide is additionally degraded into oxidised forms upon storage ofthe composition for 8 months at 30° C. after termination of themanufacturing process, e.g. freeze-drying process. In further suitableembodiments thereof, not more than about 5, 4, 3, 2, or 1.5% w/w or ofthe Factor VII polypeptide is additionally converted into oxidisedforms, as calculated from the time of termination of the manufacturingprocess until 8 months of storage at 30° C. In these embodiments of theinvention the compositions are stable such that not more than about 5%(4, 3, 2, or 1.5%) w/w of the initial content of Factor VII polypeptideis converted to oxidised forms upon storage of said composition at 30°C. for 8 months. As stated above, the initial content relates to theamount of Factor VII polypeptide added to the composition uponpreparation of the composition before the freeze-drying step.

As indicated, the degradation of Factor VII polypeptides by theaggregation pathway may also be regarded as an essential stabilityindicating parameter.

The present investigators have found that further degradation (i.e., ascalculated from the time of termination of the manufacturing processuntil 8 months of storage at 30° C.) of a Factor VII polypeptide isminimal upon storage under ambient conditions. As can be seen fromExample 6, not more than about 5% w/w, such as not more than about 4, 3,2.5, 2.0, 1.5, or 1.0% w/w of the content of a Factor VII polypeptide isadditionally recovered as aggregates upon storage for 8 months at 30° C.Also of great importance, it was found that compositions comprising asaccharide (sucrose) are more stable towards formation of aggregates.

Thus, interesting embodiments of the invention relate to compositionsthat are stable such that not more than about 5% w/w of the initialcontent of Factor VII polypeptide is converted to aggregates uponstorage of said composition at 30° C. for 8 months. As stated above theinitial content of said Factor VII polypeptide being the amount added tothe composition upon preparation of the composition before thefreeze-drying step. By proper optimisation of, at least in part, thecontents of saccharides, polyols and antioxidants, the composition isstable such that not more than about 4.0%, 3.0% w/w, such as 2.5, 2.0,1.5, or 1.0% w/w, of the initial content of Factor VII polypeptide isconverted to aggregates upon storage of said composition at 30° C. for 8months.

Thus, advantageously, the compositions of the invention have lowcontents of oxidised forms and aggregates upon termination of themanufacturing process, i.e. upon termination of the freeze-dryingprocess, and thus the compositions according to the invention arecharacterised by having a low initial content of oxidised forms andaggregates before being subjected to storage, e.g. not more than about5% w/w, such as 4%, 3%, or 2% w/w of the initial contents of Factor VIIpolypeptide is converted into an oxidised form, and less than 5% w/w,such as not more than about 4.0%, 3.0%, 2.5%, 2%, 1.5%, or not more thanabout 1% w/w, is converted into a dimeric or higher-order polymeric formupon termination of the manufacturing process

Moreover and advantageously, the compositions of the invention arestorage-stable, e.g. less than 10% w/w, such as 6%, 5%, 4%, 3%, 2%, or1.5% w/w of the initial contents of Factor VII polypeptide is convertedinto an oxidised form, and less than 5% w/w, such as 4%, 3%, 2.5%, 2%,1.5%, or 1% w/w is converted into a dimeric or higher-order polymericform upon storage at 30° C. for at least 8 months in the dark.

As mentioned, said improved stability relates to the proper combinationof particular excipients. According to the present invention, theexcipients should be selected from the group of saccharides, polyols andantioxidants in that the saccharides and polyols exhibit lyoprotectantand/or cryoprotectant properties. More specifically, in suitableembodiments according to the invention, the saccharides of interest aredi- and tri-saccharides and polysaccharides such that the saccharidesmay be selected from the group consisting of sucrose, dextrose, lactose,maltose, trehalose, cyclodextrins, maltodextrins and dextrans. Moreover,in some embodiments, the polyol is selected from the group consisting ofmannitol, sorbitol and xylitol. In still interesting embodiments, theantioxidant is selected from the group consisting of homocysteine,cysteine, cystathionine, methionine, gluthatione, and peptidescontaining any one of homocysteine, cysteine, cystathionine, methionineand gluthatione.

It is understood that the saccharide and polyol excipients,respectively, may also be a mixture of at least two such listed agents.In one series of embodiments of the invention, the saccharide excipientused is a combination of at least two di-, tri- and/or polysaccharides,such as, for example, sucrose in combination with cyclodextrin,trehalose in combination with cyclodextrin, sucrose in combination withdextran, or sucrose in combination with lactose. In one series ofembodiments of the invention, the polyol excipient used is a combinationof at least two polyols, such as, for example, mannitol in combinationwith sorbitol, mannitol in combination with xylitol, or sorbitol incombination with xylitol. In one series of embodiments of the invention,the antioxidant excipient used is a combination of at least twoantioxidants, such as, for example, methionine in combination with oneor more of homocysteine, cysteine, cystathionine, gluthatione, andpeptides containing any one of homocysteine, cysteine, cystathionine,methionine and gluthatione.

The present investigators have recognised the proper combination of thepolyols and the saccharides as well as their content so as, at least inpart, to achieve favourable stability.

Hence, in some more interesting embodiments of the invention, thepolyols are to be present in an amount ranging from about 5% w/w toabout 90% w/w. Preferably, the amount of the polyol is to be present ina range from about 18% w/w to about 88% w/w, such as from about 18% w/wto about 83% w/w, 25% to 80%, 30% to 65%, 30% to 80%, 40% to 80%, 50% to80%, 30% to 75%, 40% to 75%, 50% to 75%, or from about 50% to about 70%w/w.

The polyol are to be present in an amount ranging from about 0.5 to 75mg/ml, such as from about 2 to 60 mg/ml, 5 mg/ml to 55 mg/ml, 8 to 45mg/ml, 10 to 40 mg/ml, 10 to 30 mg/ml, or from about 2 to 45 mg/ml, 5mg/ml to 45 mg/ml, 5 to 35 mg/ml, 5 to 25 mg/ml, 5 to 20 mg/ml, 20 to 40mg/ml, or such as from about 20 to 30 mg/ml,

Moreover, in interesting embodiments thereof as well as in some otherinteresting embodiments of the invention, the saccharide is to bepresent in the composition in an amount ranging from about 0 to about85% w/w. In further interesting embodiments thereof, the amount rangesfrom about 3% w/w to about 80% w/w, such as from about 7% w/w to about75% w/w, 10% to 70%, 10% to 50%, 20% to 50%, 10% to 40%, or from about10% w/w to about 35% w/w.

The saccharide should be in an amount ranging from about 0.5 to 75mg/ml, such as from about 2 to 60 mg/ml, from about 5 mg/ml to 55 mg/ml,from about 8 to 45 mg/ml, from about 10 to 40 mg/ml, from about 10 to 30mg/ml, or from about 2 to 45 mg/ml, from about 5 mg/ml to 45 mg/ml, fromabout 5 to 35 mg/ml, from about 5 to 25 mg/ml, such as from about 5 to20 mg/ml.

Moreover, the present investigators have recognised that the properamounts of antioxidants should range between from about 0.05 to 10mg/ml, such as from about 0.1 to 5 mg/ml, 0.1 mg/ml to 2.5 mg/ml, 0.1 to2 mg/ml, or from about 0.1 to 1 mg/ml.

Importantly, the ratio between the polyol and the saccharide needs to beproper adjusted. In suitable embodiments of the invention, said polyolis in a weight ratio relative to said saccharide ranging from about100:1 to 1:50. In even more suitable embodiments thereof, said weightratio is from about 50:1 to 1:10, more preferably from about 20:1 to1:5. In other suitable embodiments, the weight ratio relates to rangesfrom about 10:1 to 1:2, and from about 6:1 to 1:2. However, as was foundout by the present investigators (see Example 5), the lyophilised cakecollapsed upon incorporating higher amounts of the saccharides. As such,very suitable embodiments relate to those wherein said sugar alcohol isin a weight ratio relative to said saccharide ranging from about 4:1 to1:1, such as from about 4:1 to 3:2 or from about 1:1 to 3:2.

In some embodiments of the invention, the polyol is mannitol and instill further embodiments the saccharide is sucrose. Moreover, in stillfurther embodiments the antioxidant is methionine.

The compositions may further be suitable formulated by incorporatingother pharmaceutically acceptable excipients so as to achievecompositions acceptable for parenteral administration, in particular tointravenous administration. Actual methods for preparing compositionsfor parenteral administration will be known or apparent to those skilledin the art and are described in more detail in for example, Remington:The Science and Practice of Pharmacy, 19th ed., Mack Publishing Company,Easton, Pa. (1995). The term “excipients” includes pharmaceuticalacceptable reagents to provide good lyophilised cake properties (bulkingagents) as well as provide lyoprotection and cryoprotection of theprotein, maintenance of pH, maintenance of acceptable tonicity as wellas proper conformation of the protein during storage so that substantialretention of biological activity and protein stability is maintained.

Thus, according to the invention, the compositions further comprise atonicity modifier. The tonicity modifier may be selected from the groupconsisting of sodium acetate, sodium lactate, sodium chloride, potassiumchloride and calcium chloride. However, other suitable tonicitymodifiers are not precluded. It is also noted, that compositions maycomprise much higher concentrations of the tonicity-modifying agent aslong as the composition is made isotonic or close to isotonic prior touse (e.g., slightly hypertonic or hypotonic), for example bulkcompositions need not to be isotonic with the physiological range.

Further stabilisation of a composition comprising a Factor VIIpolypeptide can be obtained by the addition of surfactants. Thus, instill interesting embodiments of the invention, the compositions furthercomprising a surfactant, the surfactant being selected from the groupconsisting of polysorbates, e.g. Tween®, such as polysorbate 20 or 80;polyoxyethylene alkyl ethers, e.g., polyoxyl 23 lauryl ether (Brij 35®)or poloxamers, such as poloxamer 188 (e.g. Pluronic®) or poloxamer 407,(e.g., Lutrol®) and other ethylene/polypropylene block polymers,polyethyleneglycols (PEG) such as PEG8000, or Typically, the surfactantsare added in an amount of from 0.005 to 5 mg/ml. Preferred amounts arefrom 0.01 to 3 mg/ml, more preferred from 0.01 to 0.3 mg/ml for Tween 20and/or Tween 80 and from 0.05 to 3.0 mg/ml for Poloxamer 188.

In still preferred embodiments of the invention, the composition furthercomprises other pharmaceutical excipients acting as bulking agent. Thatis to say that bulking agents other than mannitol are included in thecompositions. In particular, bulking agents are included in compositionsprepared by freeze-drying.

Initial contents of Factor VII polypeptide in the composition ispreferably from about 0.6 mg/ml to about 10.0 mg/ml, such as from about0.6 mg/ml to about 6.0 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, orfrom about 0.6 mg/ml to about 4 mg/ml.

In one embodiment, the composition comprises: Factor VII polypeptide,Mannitol, Sucrose, and Tween 80, has a moisture content of not more thanabout 3%, and has a pH in the range of 5.0 to 7.0 when the compositionis dissolved in water. In one embodiment, the composition furthercomprises one or more components selected from the list of: CaCl₂, NaCl,and Glycylglycine. In one embodiment, the Factor VII polypeptide ishuman Factor VIIa.

In one embodiment, the composition comprises: Factor VII polypeptide,Mannitol, Sucrose, methionine, and Tween 80, has a moisture content ofnot more than about 3%, and has a pH in the range of 5.0 to 7.0 when thecomposition is dissolved in water. In one embodiment, the compositionfurther comprises one or more components selected from the list of:CaCl₂, NaCl, and Glycylglycine. In one embodiment, the Factor VIIpolypeptide is human Factor VIIa.

In another embodiment, the composition comprises Factor VII polypeptide,Mannitol, Sucrose, Histidine, and Tween 80, has a moisture content ofnot more than about 3%, and has a pH in the range of 5.0 to 7.0 when thecomposition is dissolved in water. In one embodiment, the compositionfurther comprises one or more components selected from the list of:CaCl₂, NaCl, and Glycylglycine. In one embodiment, the Factor VIIpolypeptide is human Factor VIIa.

In one embodiment, the composition comprises: Factor VII polypeptide,Mannitol, Sucrose, methionine, Histidine, and Tween 80, has a moisturecontent of not more than about 3%, and has a pH in the range of 5.0 to7.0 when the composition is dissolved in water. In one embodiment, thecomposition further comprises one or more components selected from thelist of: CaCl₂, NaCl, and Glycylglycine. In one embodiment, the FactorVII polypeptide is human Factor VIIa.

In another embodiment, the composition comprises Factor VII polypeptide,Mannitol, Sucrose, and Poloxamer 188, has a moisture content of not morethan about 3%, and has a pH in the range of 5.0 to 7.0 when thecomposition is dissolved in water. In one embodiment, the compositionfurther comprises one or more components selected from the list of:CaCl₂, NaCl, and Glycylglycine. In one embodiment, the Factor VIIpolypeptide is human Factor VIIa.

In another embodiment, the composition comprises Factor VII polypeptide,Mannitol, Sucrose, methionine, and Poloxamer 188, has a moisture contentof not more than about 3%, and has a pH in the range of 5.0 to 7.0 whenthe composition is dissolved in water. In one embodiment, thecomposition further comprises one or more components selected from thelist of: CaCl₂, NaCl, and Glycylglycine. In one embodiment, the FactorVII polypeptide is human Factor VIIa.

In another embodiment, the composition comprises Factor VII polypeptide,Mannitol, Sucrose, Histidine, and Poloxamer 188, has a moisture contentof not more than about 3%, and has a pH in the range of 5.0 to 7.0 whenthe composition is dissolved in water. In one embodiment, thecomposition further comprises one or more components selected from thelist of: CaCl₂, NaCl, and Glycylglycine. In one embodiment, the FactorVII polypeptide is human Factor VIIa.

In another embodiment, the composition comprises Factor VII polypeptide,Mannitol, Sucrose, Histidine, methionine, and Poloxamer 188, has amoisture content of not more than about 3%, and has a pH in the range of5.0 to 7.0 when the composition is dissolved in water. In oneembodiment, the composition further comprises one or more componentsselected from the list of: CaCl₂, NaCl, and Glycylglycine. In oneembodiment, the Factor VII polypeptide is human Factor VIIa.

In further embodiments, the compositions are as shown in Table A below.TABLE A Formulation Formulation Formulation Formulation Compound A B C DFVII polypeptide 0.6 to 10 mg/ml 0.6 to 10 mg/ml 0.6 to 10 mg/ml 0.6 to10 mg/ml CaCl2 × 2H2O 5 to 20 mM 5 to 20 mM 5 to 20 mM 5 to 20 mM NaCl0-50 mM 0-50 mM 0-50 mM 0-50 mM Glycylglycine 0-15 mM 0-15 mM 0-15 mM0-15 mM L-Histidine 0-20 mM 0-20 mM 0-20 mM 0-20 mM Mannitol 20 to 40mg/ml 20 to 40 mg/ml 20 to 40 mg/ml 20 to 40 mg/ml Sucrose 5 to 20 mg/ml— — 5 to 20 mg/ml Methionine 0-1 mg/ml 0-1 mg/ml 0-1 mg/ml 0-1 mg/mlTween 80 0.05 to 0.15 mg/ml 0.05 to 0.15 mg/ml Poloxamer 188 — — 0.5-3mg/ml 0.5-3 mg/ml pH 5.0 to 7.0 5.0 to 7.0 5.0 to 7.0 5.0 to 7.0

In further embodiments, the compositions are as shown in Table B belowTABLE B Formulation Formulation Formulation Formulation Compound E F G HFVIIa polypeptide 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml CaCl2 × 2H2O10 mM 10 mM 10 mM 10 mM NaCl 39 mM 39 mM 39 mM 39 mM Glycylglycine 10 mM10 mM 10 mM 10 mM Mannitol 25 mg/ml 25 mg/ml 25 mg/ml 25 mg/ml Sucrose10 mg/ml 10 mg/ml 10 mg/ml 10 mg/ml Methionine 0.5 mg/ml 0.5 mg/ml 0.5mg/ml 0.5 mg/ml Tween 80 0.1 mg/ml — 0.1 mg/ml — Poloxamer 188 — 1.0mg/ml 1.0 mg/ml L-Histidine — — 10 mM 10 mM pH 6.0 6.0 6.0 6.0Formulation Formulation Formulation Formulation Compound I J K L FVIIpolypeptide 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml CaCl2 × 2H2O 10 mM10 mM 10 mM 10 mM NaCl 39 mM 39 mM 39 mM 39 mM Glycylglycine 10 mM 10 mM10 mM 10 mM Mannitol 25 mg/ml 25 mg/ml 25 mg/ml 25 mg/ml Sucrose 10mg/ml 10 mg/ml 10 mg/ml 10 mg/ml Methionine — — — — Tween 80 0.1 mg/ml —1.0 mg/ml — Poloxamer 188 — 1.0 mg/ml 1.0 mg/ml L-Histidine — — 10 mM 10mM pH 6.0 6.0 6.0 6.0

In further embodiments E1-L1, the compositions contains the excipients,and amounts thereof, listed in Table B above, but are formulated at pH5.9.

In further embodiments E2-L2, the compositions contains excipients, andamounts thereof, as listed in Table B above, but are formulated at pH5.8.

In further embodiments E3-L3, the compositions contains excipients, andamounts thereof, as listed in Table B above, but are formulated at pH 57.

In further embodiments E4-L4, the compositions contains excipients, andamounts thereof, as listed in Table B above, but are formulated at pH 56.

In further embodiments E5-L5, the compositions contains excipients, andamounts thereof, as listed in Table B above, but are formulated at pH5.5.

As discussed above, the present investigators have provided a method forstabilising Factor VII polypeptides by providing the proteins in solidcompositions comprising selected pharmaceutically acceptable excipients,of which it is important to include a stabilising agent. According tothe invention, such a stabilising agent is composed of a combination ofat least two groups of excipients selected from excipients beingsaccharides, polyols or antioxidants. Moreover, the presentinvestigators have found that saccharides (sucrose) and antioxidants(methionine) are essential for improving the stability of Factor VIIpolypeptides.

Therefore, a further aspect of the invention relates to a method ofpreparing a stable Factor VII polypeptide. The method comprises thesteps of:

i) Providing said Factor VII polypeptide in a solution comprising atleast one stability agent selected from the group consisting of

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine

ii) Processing said solution so as to obtain a solid composition with amoisture content of not more than about 3% w/w.

In particular interesting embodiments thereof, the method comprises thatsaid antioxidant is selected from the group consisting of homocysteine,cysteine, cystathionine, methionine, gluthatione, and peptidescontaining any one of homocysteine, cysteine, cystathionine, methionineand gluthatione. In a preferred embodiment, the antioxidant ismethionine. In still further interesting embodiments, said saccharide isselected from the group consisting of sucrose, dextrose, lactose,maltose, trehalose, cyclodextrins, maltodextrins and dextrans. Inpreferable embodiments the polyol is mannitol and the saccharide issucrose. Moreover, in still preferable embodiments thereof, theantioxidant is methionine.

Preferably the content of the saccharide and optionally the content ofthe antioxidant in said solution i) should be adjusted so as to achievesuperior stabilised Factor VII polypeptides.

According to the invention, the saccharide should be in an amountranging from about 0.5 to 75 mg/ml, such as from about 2 to 60 mg/ml,from about 5 mg/ml to 55 mg/ml, from about 8 to 45 mg/ml, from about 10to 40 mg/ml, from about 10 to 30 mg/ml, or from about 2 to 45 mg/ml,from about 5 mg/ml to 45 mg/ml, from about 5 to 35 mg/ml, from about 5to 25 mg/ml, such as from about 5 to 20 mg/ml.

According to the invention, the polyol should be in an amount rangingfrom about 0.5 to 75 mg/ml, such as from about 2 to 60 mg/ml, from about5 mg/ml to 55 mg/ml, from about 8 to 45 mg/ml, from about 10 to 40mg/ml, from about 10 to 30 mg/ml, or from about 2 to 45 mg/ml, fromabout 5 mg/ml to 45 mg/ml, from about 5 to 35 mg/ml, from about 5 to 25mg/ml, such as from about 5 to 20 mg/ml.

The antioxidant should be provided in an amount ranging from about 0.05to 10 mg/ml, preferably from about 0.1 to 5 mg/ml, more preferably fromabout 0.1 mg/ml to 2.5 mg/ml, even more preferably from about 0.1 to 2mg/ml, most preferably from about 0.1 to 1 mg/ml.

In a preferred embodiment, the method for preparing a stable Factor VIIpolypeptide comprises freeze-drying. The freeze-drying relates to aprocess, wherein the solution comprising said Factor VII polypeptide isfilled into lyophilisation vials or the like. Said Factor VIIpolypeptide may optionally be subjected to sterile filtration beforestart of freeze-drying. Cooling is applied to the shelves of thefreeze-drier in order to freeze the vials and the solution belowcritical product temperatures. Water is removed by introducing vacuumand condensation of water vapour on the ice-condenser of thefreeze-drier. When the product is dry, usually less than 3% residualmoisture content (e.g., measured by Karl Fischer coulometric titrationas described above), the vials are closed and capped. Manufacturing isfinalised and the composition is now in a form of a lyophilised cake.

Such compositions, when administered to a patient by injectable means,need to be reconstituted in a suitable liquid before use. They may alsobe reconstituted for other purposes, e.g. for reformulation into otherpharmaceutical compositions. However, the present invention does notpreclude that the compositions may be administered to a patient in theirsolid form.

The compositions are reconstituted using an acceptable, preferablysterile, diluent or carrier, preferably an aqueous carrier. A variety ofaqueous carriers may be used, e.g., water (e.g. Water forInjection/WFI), buffered water, saline (e.g. 0.4% saline), glycine (e.g.0.3% glycine), histidine, and the like. The reconstitution diluent mayalso contain one or more salts, such as a calcium salt (e.g. CaCl₂) or acombination of a sodium salt and a calcium salt (e.g. NaCl and CaCl₂).

The reconstituted compositions are intended for parenteraladministration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutanously, or intramuscularly, orthey are administered by way of continuous or pulsative infusion.

Therefore, a still further aspect of the invention relates to the use ofthe solid stabilised composition for the preparation of a medicament fortreating a Factor VII-responsive syndrome.

That is to say that one aspect of the invention relates to the use ofFactor VII polypeptide for the preparation of a medicament for treatinga Factor VII-responsive syndrome, said medicament comprising acomposition comprising;

a Factor VII polypeptide and a at least one stability agent selectedfrom the group consisting of

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine,

said composition having a moisture content of not more than about 3%.

Furthermore, in another aspect the invention relates to administeringsaid stabilised Factor VII polypeptide to a patient for treating aFactor VII-responsive syndrome. Thus, the invention relates to a methodof treating a Factor VII-responsive syndrome comprising administering toa subject in need thereof an effective amount of a compositioncomprising a Factor VII polypeptide, and at least one stability agentselected from the group consisting of

a) a combination of an antioxidant and mannitol;

b) a combination of methionine and a polyol;

c) a combination of a saccharide and mannitol;

d) a combination of sucrose and a polyol; and

e) methionine;

said composition having a moisture content of not more than about 3%.

In different embodiments, said Factor VII-responsive syndrome ishaemophilia A, haemophilia B, Factor XI deficiency, Factor VIIdeficiency, thrombocytopenia, von Willebrand's disease, presence of aclotting Factor inhibitor, surgery or trauma. Additionally, FactorVII-responsive syndrome may be associated with anticoagulant therapy.

As stated the composition is in solid form. Accordingly, in a suitableembodiment the medicament should be suitable for being dissolved, whichallows for parenteral administration of the medicament. Thus, whenadministering the compositions to a patient, it comprises the step ofdissolving the composition in a suitable liquid prior to theadministering step.

Abbreviations Used Herein:

FVII Coagulation Factor VII in its single chain form

FVIIa Coagulation Factor VII in its cleaved, activated two-chain form

rFVII Recombinant Factor VII (recombinant Factor VIIa)

(rFVIIa)

The following examples are offered by way of illustration, not by way oflimitation:

EXAMPLES Example 1

Compositions comprising Factor VII polypeptides and prepared accordingto Example 2 are shown. Typical excipients and their typical amounts areshown.

Table 1 shows the concentration of active ingredients and excipients inthe event where the composition is in liquid form, that is, in thecomposition before finalising the manufacturing (e.g. finalising thefreeze-drying), or in the reconstituted solution.

Table 2 shows the concentration of active ingredient and excipients inthe event where the composition is in solid form, i.e. in freeze-driedform. TABLE 1 Compositions, content of excipients in solution. Content(mg/ml) in liquid Main Function: Excipients: composition ActiveIngredient rFVIIa 0.6-5   Tonicity modifier Sodium Chloride 0-4 Tonicitymodifier/ Calcium Chloride, 2H₂0 1-7 stabiliser Buffering agentGlycylglycine 1.32 (0-1.5) Surfactant Polysorbate 80 0.05-2   BulkingAgent/ Mannitol 10-40 Cryoprotectant/ Lyoprotectant Bulking Agent/Sucrose 10-40 Cryoprotectant/ Lyoprotectant Antioxidant Methionine 0-1.0 pH 5-6

TABLE 2 Compositions, content of excipients in freeze-dried form.Content (% w/w) in solid Main Function: Excipients: composition ActiveIngredient rFVIIa 0.6-19  Tonicity modifier Sodium Chloride  0-15Tonicity modifier/ Calcium Chloride, 2H₂0 1.0 to 24.0 stabiliserBuffering agent Glycylglycine  0-6.0 Surfactant Polysorbate 80 0.05-8.0 Bulking Agent/ Mannitol 13-76 Cryoprotectant/ Lyoprotectant BulkingAgent/ Sucrose 13-76 Cryoprotetant/ Lyoprotectant Antioxidant Methionine 0-4.2 pH 5-6

Example 2

Manufacturing of Compositions

In general, the compositions were prepared from a purified bulksolution. Excipients were added, and the solution was diluted to thedesired concentration of rFVIIa. The resulting solution was sterilefiltered using a sterilised membrane filter (0.2 micron pore size orequivalent) and filled into sterile glass vials. The vials werefreeze-dried, stoppered and sealed with aluminium flip-off type caps.

Example 3

Compositions 1-19 comprising various amounts of sodium chloride,mannitol, sucrose and methionine and with various pH values wereprepared according to the process described in Example 2. Thecompositions comprised fixed concentrations of Factor VII (1.0 mg/ml),Calcium Chloride, 2H₂O (1.47 mg/ml), Glycylglycine (1.32 mg/ml) andPolysorbate 80 (1.0 mg/ml). TABLE 3 Compositions 1-19. Composition 1-19.Content of excipients mg/ml Composition No NaCl Mannitol SucroseMethionine pH 1 0 10 10 0 6.0 2 3.5 10 10 0 5.0 3 0 40 10 0 5.0 4 3.5 4010 0 6.0 5 0 10 40 0 5.0 6 3.5 10 40 0 6.0 7 0 40 40 0 6.0 8 3.5 40 40 05.0 9 0 10 10 0.5 5.0 10 3.5 10 10 0.5 6.0 11 0 40 10 0.5 6.0 12 3.5 4010 0.5 5.0 13 0 10 40 0.5 6.0 14 3.5 10 40 0.5 5.0 15 0 40 40 0.5 5.0 163.5 40 40 0.5 6.0 17 1.75 25 25 0.25 5.5 18 1.75 25 25 0.25 5.5 19 1.7525 25 0.25 5.5

Example 4

Analytical Methods Used in Determining Stability Indicating Parameters:

A. Determination of Oxidised Forms by Reverse Phase HPLC (RP-HPLC):

HPLC Column: 4.5×250 mm column packed with butylbonded silica with aparticle size of 5 μm and pore size 300 Å. Column temperature: 70° C.Eluent A: water 99.9% v/v and trifluoracetic acid 0.1% v/v. Eluent B:acetonitrile 80% v/v. trifluoracetic acid 0.09% v/v and water 19.91%v/v. The column was eluted with a linear gradient from X % B to (X+13) %B in 30 minutes. Flow rate: 1.0 ml/min. Detection: 214 nm.

The oxidised forms are methionine sulfoxides of Factor VII Polypeptides.For example the two main derivatives of FVII are Met(O)298 FVII andMet(O)306 FVII.

The content of oxidised forms is expressed as the percentage of theinitial amount of Factor VII in the composition upon preparation that isrecovered as oxidised forms of Factor VII.

B. Determination of Aggregates of Factor VII Polypeptides by HighPerformance Gel Permeation Chromatography (GP-HPLC).

GP-HPLC was run on a Waters Protein Pak 300 SW column. 7.5×300 mm. using0.2 M ammoniumsulfat pH 7.0 containing 5% isopropanol as the mobilephase. Flow rate: 0.5 ml/min and detection: 215 nm.

The content of aggregates is expressed as the percentage of the initialamount of Factor VII in the composition upon preparation that isrecovered as dimeric, oligomeric and polymeric forms of Factor VII.

Example 5

Content of Oxidised Forms of Factor VIIa After Termination of theFreeze-Drying Process and Upon Storage:

Compositions 1-19 of Example 3 were analysed by method A (Example 4) forcontent of oxidised forms upon termination of the freeze-drying and upon2 and 8 months of storage at 30° C. TABLE 4 Content of oxidised formsand increase thereof upon storage for 8 months. % of the initial contentof Factor VIIa recovered as oxidised forms. Composition increase 0-8 No0 2 8 months 1 2.5 3.5 4.8 2.3 2 2.7 3 6.1 3.4 3 2.3 3.7 7 4.7 4 2.7 5.512 9.3 5 2.6 3.6 5.3 2.7 6 2.7 3.3 5.8 3.1 7 3 3.5 5.2 2.2 8 2.3 2.9 3.41.1 9 1.6 1.5 1.7 0.1 10 1.7 1.7 2.1 0.4 11 1.7 1.8 2.5 0.8 12 1.6 1.61.9 0.3 13 1.7 1.6 1.7 0 14 1.6 1.5 1.6 0 15 1.6 1.5 1.8 0.2 16 1.7 1.61.8 0.1 17 1.7 1.6 n.a. n.a. 18 1.7 1.6 1.9 0.2 19 1.7 1.6 1.8 0.1n.a. not analysed

A statistical analysis shows that compositions comprising methionine aremore stable towards oxidative degradation.

Example 6

Content of Aggregates of Factor VIIa After Termination of theFreeze-Drying Process and Upon Storage:

Compositions 1-19 of Example 3 were analysed by method B (Example 4) forcontent of aggregates upon termination of the freeze-drying and upon 2and 8 months of storage at 30° C. TABLE 5 Content of aggregates andtheir increase upon storage for 8 months. % of the initial content ofFactor VIIa recovered as aggregates. Composition increase 0-8 No 0 2 8months 1 0.7 0.8 0.9 0.2 2 1 2 2.9 1.9 3 1.5 2.5 3.5 2 4 1.1 2.1 3.1 2 50.8 1 1.1 0.3 6 0.8 1.2 1.1 0.3 7 0.9 1 0.9 0 8 0.9 1 1.2 0.3 9 0.9 11.1 0.2 10 0.8 1.7 2.5 1.7 11 1 1.2 1.5 0.5 12 1.3 2 2.8 1.5 13 0.7 0.80.9 0.2 14 1 1.7 1.4 0.4 15 1.1 1.4 1.5 0.4 16 0.8 0.9 1 0.2 17 0.9 1.1n.a. n.a. 18 0.8 1 1.5 0.7 19 1 1 1.1 0.1n.a. not analysed

A statistical analysis shows that the presence of sucrose in thecompositions is important in order to decrease formation of aggregates.

Example 7

Content of Dimeric and Oligomeric Forms of Factor VIIa After Terminationof the Freeze-Drying Process:

Composition A comprising polyol and saccharide as listed in the tablebelow was prepared according to the process described in Example 2. Thecomposition further comprised Factor VIIa (1.0 mg/ml), calcium chloride2H₂O (1.47 mg/ml), glycylglycine (1.32 mg/ml) and polysorbate 80 (0.7mg/ml). pH was 5.0.

Composition A was analysed by method B (Example 4) for content ofaggregates upon termination of the freeze-drying and upon 1 and 2 monthsof storage at 30° C. The table lists the % of the initial content ofFactor VIIa recovered as aggregates: TABLE 6 % of the initial content ofFactor VIIa recovered as aggregates. Months Composition Additives 0 1 2A Mannitol 25 mg/ml 1.1 2.0 1.7 Trehalose 15 mg/ml

Example 8

Structural stability of the freeze-dried cake is shown for variouscompositions, 1-19, see Example 3. The ratio of mannitol to sucrose isreported of each composition. TABLE 7 Stability of the freeze-dried cakeaccording to the ratio between mannitol and sucrose Compositions 5, 6,1, 2, 7, 8, 9, 17, 18 3, 4, 13, 14 10, 15, 16 and 19 11, 12 Weight Ratio1:4 1:1 1:1 4:1 of Man:Suc Freeze-dried C OK OK OK cakeC: collapsed cake,Man: Mannitol,Suc: Sucrose,OK: slightly collapsed or not collapsed

Example 9

The following formulations were prepared as described in example 2 usingfilling volumes of 1 and 5 ml: Formulation A B C rFVIIa (mg/ml) 0.6 1.01.0 CaCl₂ (mM) 10 10 10 Glycylglycine 10 10 10 L-Histidine (mM) — 10 10NaCl (mM) 50 39 39 Tween 80 (mg/ml) 0.038 0.07 0.07 Methionine (mg/ml) —0.5 2.0 Mannitol (mg/ml) 30 25 25 Sucrose (mg/ml) — 10 10 Ph 5.5 5.5 5.5

The stability of the formulations was investigated at 25° C., 30° C.,and 40° C. and the following results were obtained. The vials werereconstituted in water before analysis.

Dimer and Oligomer Forms

The contents of dimer and oligomer forms were measured by GP-HPLC asdescribed in example 4. All results are stated in %.

5 ml Filling Volume: ½ 3 months 6 Formula- 0 month 1 month 40° 30° 25°months tion months 40° C. 40° C. C. C. C. 25° C. A 3.3 5.5 6.4 8.0 6.06.3 6.3 B 2.3 2.7 2.8 3.0 2.7 3.2 3.0 C 4.0 4.2 4.6 4.7 4.3 4.4 4.4

1 ml Filling Volume: 3 months Formulation 0 months 40° C. 30° C. 25° C.A 2.7 11.0 7.0 6.5 B 2.2 7.4 3.4 2.9 C 3.3 6.9 3.8 3.7

Furthermore, the contents of aggregate forms for formulation A (=Ref)and formulation B (=New), filling volume 5 ml, can be seen from FIG. 2a.

Oxidised Forms

The contents of oxidised forms were measured by RP-HPLC as described inexample 4.

5 ml Filling Volume: ½ 3 months 6 Formula- 0 month 1 month 40° 30° 25°months tion months 40° C. 40° C. C. C. C. 25° C. A 2.1 2.8 3.4 4.0 3.43.1 3.5 B 1.3 1.3 1.6 1.3 1.6 1.6 1.4 C 1.2 1.4 1.6 1.8 1.6 1.6 1.3

1 ml Filling Volume: 3 months Formulation 0 months 40° C. 30° C. 25° C.A 2.5 4.6 4.0 5.3 B 1.3 2.0 1.1 1.8 C 1.3 1.8 1.7 1.5

Furthermore, the contents of oxidized forms for formulation A (=Ref) andformulation B (=New), filling volume 5 ml, can be seen from FIG. 2 b.

Activity

The activity was measured by one stage clotting assay as described inexample 12 for formulations, which had been stored at 30° C. for 3months. The specific activity was calculated based on the content ofrFVIIa in the formulations and the following results were obtained:Specific activity Formulation (IU/mg) A 50523 B 58373 C 60318

Interpretation of Results:

The results show, that formulations B and C, which contain mannitol,sucrose, and methionine are more stable—i.e. shows a slower increase incontents of dimer and oligomer forms and oxidised forms. In accordance,the activities of these formulations are higher as measured after 3months' storage at 30° C.

Example 10

The following formulations were prepared as described in example 2 usinga filling volume of 5 ml. Formulation A B C D E rFVIIa (mg/ml) 1.0 1.01.0 1.0 1.0 CaCl₂ (mM) 10 10 10 10 10 Glycylglycine (mM) 10 10 10 10 10L-Histidine (mM) 10 10 10 10 10 NaCl (mM) 39 39 39 39 39 Methionine(mg/ml) 0.5 0.5 0.5 0.5 0.5 Tween 20 (mg/ml) 0.1 Tween 80 (mg/ml) 0.1Poloxamer 188 (mg/ml) 1.0 Brij 35 (mg/ml) 0.1 Mannitol (mg/ml) 25 25 2525 25 Sucrose (mg/ml) 10 10 10 10 10 pH 5.5 5.5 5.5 5.5 5.5

The vials were reconstituted in water and subjected to shaking for 19hours in order to test the physical stability of the formulations. Theeffect of shaking was investigated by visual inspection and measurementof the UV absorbance at 400 nm (=Abs in the table below). The resultsbelow clearly show the increased physical stability obtained by additionof a detergent. Before shaking Visual After shaking Formulationappearance Abs Visual appearance Abs A Clear liquid 0.01 Clear liquidwith few particles 0.01 B Clear liquid 0.00 Slightly turbid 0.36 C Clearliquid 0.00 Clear liquid with few particles 0.00 D Clear liquid 0.00Clear liquid with few particles 0.00 E Clear liquid 0.01 Turbid 2.18

Example 11

Six formulation were prepared as described in example 2. The compositionof the formulations was: rFVIIa 1.0 mg/ml CaCl₂ 10 mM Glycylglycine 10mM L-Histidin 10 mM NaCl 39 mM Tween 80 0.07 mg/ml Mannitol 25 mg/mlSucrose 10 mg/ml

Each of the six formulations were adjusted to a different pH:

Formulation A: pH 5.0

Formulation B: pH 5.5

Formulation C: pH 6.0

Formulation D: pH 6.5

Formulation E: pH 7.0

Formulation F: pH 7.5

The stability of the six formulations was investigated for vials storedat 25° C. and 40° C. The results shown in the Tables below (and FIGS. 1a and 1 b) were obtained. Dimer and oligomer forms (aggregates) (%)Formulation 0 MONTHS 40° C./3 MONTHS 25° C./3 MONTHS A 2.2 3.8 2.5 B 1.73.2 1.9 C 1.5 2.7 1.7 D 1.1 1.9 1.3 E 0.8 1.6 1.0 F 0.8 1.6 1.0

Oxidised forms (%) Formulation 0 MONTHS 40° C./3 MONTHS 25° C./3 MONTHSA 2.7 3.3 3.4 B N/A 4.0 3.6 C 2.6 5.0 3.7 D 2.5 4.9 3.6 E 2.6 10.0 4.6 F2.6 8.7 4.7

The contents of dimers and oligomer forms and oxidised forms weredetermined as described in example 4.

Example 12

Assays for Testing Biological Activity of Factor VII Polypeptides:

Test for Factor VIIa Activity:

A suitable assay for testing for Factor VIIa activity and therebyselecting suitable Factor VIIa variants can be performed as a simplepreliminary in vitro test: (the “In Vitro Hydrolysis Assay”).

In Vitro Hydrolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) may be assayed for specific activities.They may also be assayed in parallel to directly compare their specificactivities. The assay is carried out in a microtiter plate (MaxiSorp,Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide(S-2288, Chromogenix, Sweden), final concentration 1 mM, is added toFactor VIIa (final concentration 100 nM) in 50 mM Hepes, pH 7.4,containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during a20-minute incubation, after subtraction of the absorbance in a blankwell containing no enzyme, is used to calculate the ratio between theactivities of variant and wild-type Factor VIIa:Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Based thereon, Factor VIIa variants with an activity comparable to orhigher than native Factor VIIa may be identified, such as, for example,variants where the ratio between the activity of the variant and theactivity of native Factor VII (wild-type FVII) is around, versus above1.0.

The activity of Factor VIIa or Factor VIIa variants may also be measuredusing a physiological substrate such as Factor X, suitably at aconcentration of 100-1000 nM, where the Factor Xa generated is measuredafter the addition of a suitable chromogenic substrate (eg. S-2765)(“the In Vitro Proteolysis Assay”). In addition, the activity assay maybe run at physiological temperature.

In Vitro Proteolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage is then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mlbovine serum albumin. The amount of Factor Xa generated is measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used to calculate the ratio between the proteolyticactivities of variant and wild-type Factor VIIa:Ratio=(A405 nm Factor VIIa variant)/(A405 nm Factor VIIa wild-type).

Based thereon, Factor VIIa variants with an activity comparable to orhigher than native Factor VIIa may be identified, such as, for example,variants where the ratio between the activity of the variant and theactivity of native Factor VII (wild-type FVII) is around, versus above1.0.

Thrombin Generation Assay:

The ability of Factor VII or Factor VII-related polypeptides or FactorVIII or Factor VIII-related polypeptides (e.g., variants) to generatethrombin can be measured in an assay comprising all relevant coagulationFactors and inhibitors at physiological concentrations and activatedplatelets (as described on p. 543 in Monroe et al. (1997) Brit. J.Haematol. 99, 542-547 which is hereby incorporated as reference).

Clot Assay:

The activity of the Factor VII polypeptides may also be measured using aone-stage clot assay (assay 4) essentially as described in WO 92/15686or U.S. Pat. No. 5,997,864. Briefly, the sample to be tested is dilutedin 50 mM Tris (pH 7.5), 0.1% BSA and 100 μL is incubated with 100 μL ofFactor VII deficient plasma and 200 μL of thromboplastin C containing 10mM Ca²⁺. Clotting times are measured and compared to a standard curveusing a reference standard or a pool of citrated normal human plasma inserial dilution.

1. A composition comprising a Factor VII polypeptide, and at least onestability agent selected from the group consisting of a) a combinationof an antioxidant and mannitol; b) a combination of methionine and apolyol; c) a combination of a saccharide and mannitol; d) a combinationof sucrose and a polyol; and e) methionine, said composition having amoisture content of not more than about 3%.
 2. The composition accordingto claim 1, wherein the composition comprises a combination of anantioxidant and mannitol and further comprises a saccharide.
 3. Thecomposition according to claim 1, wherein the composition comprises acombination of methionine and a polyol and further comprises asaccharide.
 4. The composition according to claim 1, wherein thecomposition comprises a combination of a saccharide and mannitol andfurther comprises an antioxidant.
 5. The composition according to claim1, wherein the composition comprises a combination of sucrose and apolyol and further comprises an antioxidant.
 6. The compositionaccording to claim 1, wherein the antioxidant is selected from the groupconsisting of homocysteine, cysteine, cystathionine, methionine,gluthatione, and peptides containing any one of homocysteine, cysteine,cystathionine, methionine, and gluthatione.
 7. The composition accordingto claim 1, wherein the saccharide is selected from the group consistingof sucrose, dextrose, lactose, maltose, trehalose, cyclodextrins,maltodextrins and dextrans.
 8. The composition according to claim 1,wherein the polyol is selected from the group consisting of mannitol,sorbitol and xylitol.
 9. The composition according to claim 1, whereinthe composition is stable such that not more than about 5% w/w of theinitial content of Factor VII polypeptide is converted to aggregatesupon storage of said composition at 30° C. for 8 months.
 10. Thecomposition according to claim 1, wherein the composition is stable suchthat not more than about 6% w/w of the initial content of Factor VIIpolypeptide is converted to oxidised forms upon storage of saidcomposition at 30° C. for 8 months.
 11. The composition according toclaim 1, wherein said polyol is in an amount ranging from about 5% w/wto 90% w/w.
 12. The composition according to claim 1, wherein saidsaccharide is in an amount ranging from about 0 to 85% w/w.
 13. Thecomposition according to claim 1, wherein said polyol is in a weightratio relative to said saccharide ranging from about 100:1 to 1:50. 14.The composition according to claim 1, further comprising an agentsuitable for keeping the pH of said composition in the range of 3 to 9when dissolved in aqueous solvent.
 15. The composition according toclaim 14, wherein said agent is selected from the group consisting ofcitrate, acetate, histidine, malate, phosphate, tartaric acid, succinicacid, MES, HEPES, PIPES, imidazol, TRIS, lactate, glutamate andglycylglycine.
 16. The composition according to claim 1, furthercomprising a tonicity modifier.
 17. The composition according to claim16, wherein the tonicity modifier is selected from the group consistingof sodium acetate, sodium lactate, sodium chloride, potassium chlorideand calcium chloride.
 18. The composition according to claim 1, furthercomprising a surfactant.
 19. The composition according to claim 18,wherein the surfactant is selected from the group consisting ofpolysorbates; polyoxyethylene alkyl ethers; poloxamers;ethylene/polypropylene block polymers; and polyethyleneglycols (PEGs).20. The composition according to claim 1, further comprising one or moreother pharmaceutical excipients acting as a bulking agent.
 21. Thecomposition according to claim 1, wherein the saccharide is sucrose. 22.The composition according to claim 1, wherein the polyol is mannitol.23. The composition according to claim 1, wherein the Factor VIIPolypeptide is selected from the group consisting of Human Factor VIIaand a Factor VII Sequence Variant.
 24. The composition according toclaim 23, wherein the Factor VII Polypeptide is recombinant human FactorVIIa.
 25. The composition according to claim 1, wherein the Factor VIIPolypeptide is a Factor VII-related polypeptide and wherein the ratiobetween the activity of said Factor VII-related polypeptide andwild-type Factor VII is at least 1.25 when tested in an in vitroproteolysis assay or in an in vitro hydrolysis assay.
 26. Thecomposition according claim 23, wherein Factor VII polypeptide ispresent in a concentration of from about 0.6 mg/ml to about 10.0 mg/ml.27. The composition according to claim 1, wherein said moisture contentis not more than about 2.5% w/w.
 28. The composition according to claim1, wherein the composition is a lyophilised cake.
 29. The compositionaccording to claim 1, wherein the composition comprises: Factor VIIpolypeptide, Mannitol, Sucrose, and a surfactant selected from the groupconsisting of a polysorbate and a poloxamer.
 30. The compositionaccording to claim 29, further comprising methionine.
 31. Thecomposition according to claim 29, further comprising L-histidine. 32.The composition according to claim 29, further comprising one or morecomponents selected from the group consisting of: CaCl₂, NaCl, andGlycylglycine.